The present invention relates to novel compositions that are effective in inhibiting apoptotic cell death and to methods of preparing and using such compositions.
Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging.
Generally, cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane, permeability and intemucleosomal DNA cleavage characterize apoptotic cell death. Specific intemucleosomal DNA fragmentation is a hallmark for many, but notably not all, instances of apoptosis. Apoptotic cell death appears to play a significant role in the tissue damage that occurs in association with various abnormal conditions, for example, ischemia, various gastrointestinal disorders, and organ transplantation.
Ischemia is the result of deceased blood flow to a particular area or organ of the body. Ischemia is responsible for several important types of physiologic damage such as brain damage, spinal cord trauma and myocardial ischemia. For example, recent data indicate that apoptosis plays a significant role in heart injury induced by ischemia and subsequent reperfusion. Severe cell damage during prolonged ischemia appears to result in necrotic death of myocardial cells. However, if the ischemia is relatively limited in extent and duration, the apoptotic pathway is initiated. Although early reperfusion decreases heart damage, massive cell death can occur with the restoration of blood flow. In this instance, the cells that die are those that remained viable at the end of ischemia Therefore, restoration of blood flow allows apoptosis to proceed. IGF and Calpain inhibitors, which are capable of preventing apoptosis in different systems, also inhibited apoptosis of cardiomyocytes following ischemia and reperfusion both in vito and in vitro.
As noted above, apoptosis has also been associated with various gastrointestinal disorders, including those caused by anticancer therapies. Gastrointestinal toxicity of anticancer therapies has been associated with the alteration of DNA metabolism and cell replication. In cancer patients, chemotherapy and radiotherapy damage cells and alter DNA metabolism and replication. These treatments not only kill rapidly dividing tumor cells, but also healthy stem cells in the intestinal crypts because of their rapid division. Destruction of mucosal cells by anti-cancer agents results in mucositis, stomatitis, diarrhea, decreased nutrient absorption, bacterial infections and anorexia. The dose of chemotherapy or radiation is often limited by these side effects.
Cancer patients can suffer from severe metabolic changes and mental stress resulting in cachexia and appetite loss. Common chemotherapy regimens can worsen these gastrointestinal disorders even further causing nausea and direct damage to epithelial cells. Careful consideration of the diet of patients can alleviate some of the destructive effects of chemotherapy. A variety of food supplements containing, in part, partially processed plant extracts have been used to ameliorate the gastrointestinal disorders that often accompany chemotherapy, radiation and AIDS. The supplements generally contain carbohydrates, fat and plant protein hydrolysates. Tomei and Cope et al., Apoptosis The Molecular Basis of Cell Death (Cold Springs Harbor Laboratory Press, 1991).
As shown in rat models, methotrexate treatment causes alterations of gastrointestinal mucosa leading to severe diarrhea, loss of appetite, and loss of weight in rats fed diets containing casein as the sole protein source. Previous experiments have demonstrated that rats fed a diet containing soy flour or isolated soy products experience a reduction in the incidence of these undesirable side-effects (Chevreau and Funk-Archuletta, Toxicity of Chemotherapy. pp. 269-295 (Grune and Stratton, N.Y., 1984); Funk and Baker, J. Nutr., 121:1684-1692 (1991); Funk and Baker, J. Nutr., 121:1673-1683 (1987)). A previously described anti-apoptotic fraction isolated from soy flour, referred to as soy-derived anti-apoptotic fraction (SDAAF), also referred to as AcE, for example, in U.S. Pat. No. 5,635,186), proved to be effective in alleviating methotrexate toxicity, which was confirmed by improved food intake, weight gain and absence of the diarrhea commonly observed following methotrexate injection in control animals fed casein based diets (Funk-Archuleta et al., Nutrition and Cancer, 29(3):217-221 (1991)).
The soy-derived anti-apoptotic fraction (SDAAF) reduces methotrexate toxicity likely by reduction of apoptosis (programmed cell death) (Funk-Archuletta et al., Nutrition and Cancer, supra). Methotrexate, similar to other chemotherapeutic agents and radiation treatment, increases the incidence of apoptosis in the areas of rapidly dividing cells, including gastrointestinal tract mucosa. Results from CX3H10Txc2xd cell-based assay determined that SDAAF is a potent inhibitor of apoptosis (Funk-Archuletta et al., Nutrition and Cancer, supra, Tomei, et al., Proc. Nat""l Acad. Sci (USA), 90:853-857 (1993)). It has also been reported that hydrolyzed soy protein products can decrease radiation-induced apoptosis in intestinal epithelial cells (Cope et al., FASEB J., 5:a931 (1991)).
Plant-derived delipidated extracts that produce an anti-apoptotic effect are described in PCT Publication No. WO 95/15173, U.S. Pat. No. 5,567,425 and U.S. Pat. No. 5,635,186. These extracts, referred to as xe2x80x9cAcExe2x80x9d in these references and as soy-derived anti-apoptotic fraction (SDAAF) herein, contain the phospholipids lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylinositol (LP), phosphatidic acid (PA) and phosphatidylinositol (PI) in addition to various protein and carbohydrate constituents.
Phospholipids are a class of amphipathic phosphorous-containing lipids that are essential constituents of biological membranes. Various phospholipid preparations have been used for cooking, drug delivery (liposomes), slow release delivery systems, carrier media for hydrophobic drugs, gene transfer and replacement therapy, sunscreens, emulsions, anti-foaming agents, replacement of damaged or absent pulmonary surfactants, detergents and membrane stabilization. PA, PI, LPC, LPI, and LPA are found in a variety of plant and animal products. LPA has been reported to have a variety of physiological activities including mitogenesis, growth factor, wound healing and as an anti-wrinkle agent. U.S. Pat. Nos. 4,263,286; 4,746,652; 5,326,690; 5,480,877; and 5,340,568. LPA is reviewed in detail by Moolenaar (1994) TICB 4:213-219; Eichholtz et al. (1990) Biochem. J. 291:677-680; and Moolenaar (1995) J. Biol. Chem. 270:12949-12952.
Several proteinase inhibitors derived from plant extracts have anticarcinogenic activity, including the Bowman-Birk Inhibitor. Birk (1985) Int. J. Pep. Pro. Res. 25:113-131. The Bowman-Birk Inhibitor is a disulfide bonded protein with a molecular weight of about 8 kD that can inhibit the activity of trypsin and chymotrypsin. This inhibitor has been found in crude soybean extracts and has been characterized immunologically. The Bowman-Birk Inhibitor has been found to suppress cellular transformation and have activity in degranulation of macrophages. The inhibitor is also known to increase pancreas size when fed to animals. The Bowman Birk Inhibitor, however, lacks anti-apoptotic activity.
In contrast, the compositions obtained from plant-derived extracts as described, for example, in U.S. Pat. No. 5,635,186, have anti-apoptotic activity. Accordingly, these compositions are unrelated to the Bowman Birk Inhibitor.
These anti-apoptotic compositions were generally prepared by extracting the aqueous solution from delipidated soybean powder to obtain an aqueous retentate in which the anti-apoptotic activity is soluble in a mixture of aqueous and organic solvent as described in U.S. Pat. No. 5,635,186. Although this earlier method is adequate, the present invention provides more efficient and cost effective methods of preparing compositions having anti-apoptotic activity.
The present invention provides methods for the aqueous extraction of anti-apoptotic compositions from protein isolates or lipid mixtures. The methods are generally accomplished by first mixing a protein isolate, a lipid mixture or a mixture thereof in an aqueous, non-organic solvent containing solution, and thereafter separating the aqueous solution from the insoluble material. Since the anti-apoptotic composition is soluble in the aqueous solution, the methods of the present invention provides a simple water extraction of the desired anti-apoptotic composition from the insoluble material. The extracted anti-apoptotic compositions can then be isolated from the aqueous solution by any means known in the art and, if desired, dried for various purposes.
Protein isolates useful as starting materials in the methods can be derived from plants, plant organs or plant extracts. Particularly suitable protein isolates are derived from plants of the leguminosae, solanum and allium families, especially soybeans. Useful lipid mixtures can be isolated from plant and animal tissues containing phospholipids or phosphatides, with one of the several lecithins being particularly useful, especially soybean-derived lecithins. Additionally, combinations of such protein isolates and lipid mixtures are useful in obtaining the anti-apoptotic compositions of the present invention. A ratio of 4:1 protein isolate to lipid mixture is particularly useful in the methods.
The present invention further provides the anti-apoptotic compositions produced by the water-extraction methods. The novel compositions can be used to treat or prevent an adverse condition associated with apoptosis, including, for example, gastrointestinal disorders, deleterious dermatological conditions, immunosuppression or immunodeficiency, reperfusion damage resulting from ischemia, cardiovascular disorders, transplantation, would healing, tissue rejection and Alzheimer""s disease.